RNA干扰技术抑制宫颈癌HeLa细胞IKCa1表达的探究.pdfVIP

《ffii．；●I●1； RNA干扰技术抑制宫颈癌HeLa细胞IKCat表达的研究 摘 要 计两条针对IKCal基因的干扰序列和一条非特异性阴性对照序列，并将其 异性阴性对照序列)，转化感受态细菌DH5a，培养后提取质粒；采用酶切、 测序鉴定所提取质粒。利用脂质体法将构建的质粒转染入宫颈癌HeLa细 mRNA 总RNA，以荧光定量PCR方法检测转染前后HeLa细胞株IKCal 的变化。转染后48h提取各组细胞的总蛋白，以Western Blotting法检测转 细胞增殖情况并绘制生长曲线。用流式细胞术分析细胞周期变化并计算细 胞增殖指数PI。结果：经酶切和测序鉴定显示IKCalshRNA真核表达载体 mRNA表 达。Western 性(PO．05)：结论：所构建的IKCal 制细胞增殖，使细胞周期停滞于G(舳l期。 关键词：子宫颈癌；HeLa细胞株；钙激活性中电导钾离子通道； RNA干扰；荧光定量PCR；Western I流式细胞术， Blotting；WST．I that one control were to of designed negative oligonucleotide referring double-strandedRNAwasderived GenBank，then through clonedinto vectordirectedorientation．The vector pGenesil一1．1 pGenesil一1．1 hasbeen EGFETherecombinantwastransformedintothe signedby plasmid DH5a．The wascultured LBmediumto competencegerm germ byusing extractthe whichWasincisivedand Wasdone plasmid sequenced．Transfection withthe of 2000．The ofIKCalmRNAwas help lipofectarnineexpression detectedwith the of FQ-PCR(fluorescencequantitativePCR)andexpression IKCal withwestern was wasdetected protein blotting．Cellproliferation detectedwithWST-1 wasdetectedwithflow assay．Cell